Mitochondrial complex I (NADH-ubiquinone reductase) bears coupling site I, and is composed of lesser than 25 unlike polypeptides, FMN and 8 iron-sulfur (FeS) clusters. Four FeS clusters are rapidly reduced by NADH, one has an Em lesser than -400 mV, and three are EPR silent. The polypeptides containing FMN and FeS clusters are encased within lesser than 17 hydrophobic polypeptides. The complexity of mammalian complex I has hampered progress in study of many aspects of its structure and function. The respiratory chain of aerobically grown Paracoccus denitrificans contains coupling site I, is inhibited by rotenone, and exhibits EPR signals similar to those of mitochondrial complex I. The cytochrome oxidase of this organism is also similar to the mitochondrial enzyme in terms of electron carriers (cytochromes aa3, 2Cu). However, the P. denitrificans oxidase is composed of only 2 polypeptides, while the mammalian enzyme contains 7 major and possibly 3 to 5 minor polypeptides. The purpose of this project is to isolate complex I from P. denitrificans and study its structure; polypeptide, flavin and FeS composition; and mechanisms of electron transfer and H+ translocation. Specific plans are: (a) Isolation from P. denitrificans of a rotenone-sensitive NADH-Q reductase; (b) analysis of the complex for number of polypeptides, and concentrations of flavin (also whether FMN or FAD); Fe, S2-, Q, phospholipids; (c) EPR studies of the FeS clusters and their Em; (d) resolution of the complex by chaotropes for possible isolation of flavoprotein and FeS protein fragments, and their analysis as in b to d; (e) studies with surface-labeling reagents to identify accessible polypeptides; (f) production of antibodies to the complex and resolved fragments, and their use for functional, topological and membrane orientation studies; (g) reconstitution of chaotrope-resolved fragments to understand electron transfer sequence and inhibition sites of rotenone, etc.; (h) use of protein residue modifiers for specific inhibition and possible discrimination between electron transfer and H+ translocation, and identification of components involved in H+ translocation; (i) isolation (directly or by immunoprecipitation) of the enzyme from rotenone-insensitive mutants or cells grown under sulfate-limited conditions. These membranes lack coupling site I and EPR signal due to FeS cluster 2. Comparative structural-functional studies with normal complex I should shed light on the mechanisms of H+ translocation at site I.